DuraniMagliery2012ProteinExprPurif[1]

ReviewSimplifying protein expression with ligation-free,traceless and tag-switching plasmidsVenuka Durani a ,Brandon J.Sullivan b ,Thomas J.Magliery a ,c ,⇑aDepartment of Chemistry,The Ohio State University,Columbus,OH 43210,USAbOhio State Biochemistry Program,The Ohio State University,Columbus,OH 43210,USA cDepartment of Biochemistry,The Ohio State University,Columbus,OH 43210,USAa r t i c l e i n f o Article history:Received 17March 2012and in revised form 1June 2012Available online 21June 2012Keywords:PlasmidProtein expression Co-expression Protease cleavageLigation independent cloning Traceless tagginga b s t r a c tSynthetic biology and genome-scale protein work both require rapid and efficient cloning,expression and purification.Tools for co-expression of multiple proteins and production of fusion proteins with purifica-tion and solubility tags are often desirable.Here we present a survey of plasmid vectors that provide for some of these features with a focus on tools for rapid cloning and traceless tagging –a setup that facil-itates removal of fusion tags post-purification leaving behind no ‘scar’on the final construct.Key features are reviewed,including plasmid replication origins and resistance markers,transcriptional promoters,cloning methods,and fusion tags and their removal by proteolysis.We describe a vector system called pHLIC,which assembles features for simple cloning,overexpression,facile purification,and traceless cleavage,as well as flexibility in modifying the vector to exchange fusion tags.Ó2012Elsevier Inc.All rights reserved.ContentsIntroduction.............................................................................................................9Origin of replication .....................................................................................................10Antibiotic resistance .....................................................................................................10Cloning regions .........................................................................................................10Transcription promoters..................................................................................................12Fusion tags.............................................................................................................13Protease cleavage sites ...................................................................................................13Seamless cloning and traceless tagging.. (13)Vectors for traceless tagging –some examples............................................................................13pHLIC vectors ......................................................................................................14Conclusion.............................................................................................................15Appendix A.Supplementary data . (15)References (15)IntroductionHigh-throughput approaches in modern protein science –including structural genomics and proteomics –have necessitated the development of high-throughput methods for cloning,protein expression,and purification [1–5].Even for laboratories studying a single protein target,such as designed proteins,these steps are often expensive and time-consuming prerequisites,particularly for beginning researchers.Solutions to these problems have emerged [6–9]:recombinogenic (e.g.,Gateway [10])cloning and ligation-independent cloning (LIC)1[11,12];vectors with very1046-5928/$-see front matter Ó2012Elsevier Inc.All rights reserved./10.1016/j.pep.2012.06.007⇑Corresponding author at:Department of Chemistry,The Ohio State University,Columbus,OH 43210,USA.Fax:+16142921685.E-mail addresses:vdurani@ (V.Durani),bsulliva@ (B.J.Sullivan),magliery.1@ (T.J.Magliery).1Abbreviations used:MBP,maltose binding protein;GST,glutathione S-transferase;CBP,calmodulin binding protein;Trx,thioredoxin;PSI–MR,protein structure initiative–materials repository;LIC,ligation independent cloning;SLiCE,seamless ligation cloning extract;IPTG,isopropyl b -D -1-thiogalactopyranoside;IMAC,immo-bilized metal-ion affinity chromatography;SUMO,small ubiquitin-like modifier;Ub,ubiquitin;DUBs,deubiquitylating enzymes;TEV,tobacco etch virus;TIM,triose-phosphate isomerase.strong promoters such as T7and cspA ;and the use of fusion tags such as hexahistidine (6ÂHis)[13],maltose binding protein (MBP)[14,15],glutathione S-transferase (GST)[16,17],calmodu-lin-binding peptide (CBP)[18,19]and thioredoxin (Trx)[20]for solubility and/or purification.Many useful vectors are available through the Protein Structure Initiative Material Repository (PSI–MR),and information about these vectors is available in a search-able database called DNASU [7].While affinity tagging results in convenient purification,many applications demand removal of the fused tag post-purification,typically by proteolysis.However,the recognition sequences of restriction enzymes for cloning and of proteases for freeing the fi-nal protein typically put constraints on one or more amino acids in the protein,leaving a ‘scar’that may not be desirable.This problem has initiated work on seamless cloning and traceless tagging strat-egies,and plasmid vectors that simplify such manipulations are desirable.Plasmid vectors require various components to carry out their functions (Fig.1).The origin of replication determines the copy number of a plasmid;antibiotic resistance provides a selection marker;promoter regions facilitate gene transcription;and careful engineering of cloning sites can provide for features like ligation-independent cloning and fusion tags to facilitate solu-bility and purification of the expressed protein.Vectors with un-ique combinations of these functionalities are especially suited for specific applications in structural genomics and synthetic biol-ogy.Here we present a brief survey of plasmid vectors useful for protein expression with a focus on vectors that facilitate quick cloning and traceless tagging of proteins.Origin of replicationThe origin of replication is the smallest region of the plasmid that is able to replicate on its own and is also the site where plas-mid replication is initiated [21].The replicator region of a plasmidthat includes the origin of replication determines its copy ually for large scale protein purification we turn to high copy number plasmids that can maintain tens to hundreds of copies of plasmid per cell.Production of recombinant proteins in Escherichia coli often results in degradation,aggregation or misfolding of pro-teins,and co-expression [22]of molecular chaperones can provide a solution to this problem [23–25].Co-expression of proteins is also desirable for purification of multi-subunit complexes and studying interacting proteins [26–28].However,plasmid incom-patibility,a mechanism that prevents the stable co-existence of two similar plasmids in the same bacterial cell,can cause problems for these kinds of applications,and having access to plasmids with different origins of replication that are compatible with each other is important.The best developed class of plasmid vectors for many manipulations including protein expression is based on the ColE1/pMB1[29]replicons including the pBR322[30,31],pUC [32,33]and pET (Novagen)vector systems.Vectors containing ColE1/pMB1de-rived origins of replication fall into the same incompatibility group [34,35].On the other hand,plasmids like pACYC177and pACYC184[36,37]with origins of replication derived from p15A [38],although incompatible with other vectors with p15A derived ori-gins,are compatible with ColE1/pMB1derived plasmids.These are two compatible vector groups that are commonly used for co-expression of proteins.Since ColE1based plasmids are very common for production of target proteins,several chaperone-encoding plasmids are available on vectors with p15A derived rep-licons [23].For experiments in which the population of three or more different plasmids needs to be maintained in a cell,other plasmids with pSC101[39],CloDF13[40,41],ColA [42,43],RF1030[44]and pEC [45]replicons can be used [22].Antibiotic resistanceIn order to ensure that a plasmid vector is taken up and main-tained in a cell,genes encoding resistance to antibiotics are ex-pressed from plasmids.Moreover,if populations of multiple vectors are to be maintained in a cell,then not only do their repli-cator regions need to be compatible,their antibiotic resistance markers also need to be orthogonal to ensure co-transformation.The most common antibiotic resistance markers are ampicillin,kanamycin,chloramphenicol and tetracycline.Table 1has exam-ples of some vectors with spectinomycin,zeocin and erythromycin resistance.Ampicillin is especially susceptible to degradation when present the bacterial culture and this effect can be observed as ‘satellite’colonies on agar plates.This effect can be alleviated to some extent by using carbenicillin [46],a less degradation-susceptible analog of ampicillin.Cloning regionsMost plasmid vectors that are commercially available have multiple unique restriction enzyme sites arranged in tandem in a polylinker cloning region.These can be used for traditional liga-tion-dependent cloning methods (Fig.2a)where a target gene is amplified by PCR using primers containing unique restriction sites,followed by restriction enzyme digest and ligation into a vector containing compatible cohesive ends.These traditional methods often produce ‘background’due to incomplete digestion and re-ligation of the digested vector and require unique restriction en-zyme sites in the vector that do not occur in the DNA to be cloned.These limitations and others such as difficulty to manipulate very large inserts have led to research in recombinogenic cloning meth-ods especially for applications in structural genomics.Homologous recombination occurs through stretches of DNA shared by the two molecules.Because the sequence of the homology regions canbe1.Schematic plasmid map (not drawn to scale)showing various important regions of plasmid vectors that are discussed in this review.and Purification 85(2012)9–17chosen freely,any position on a target molecule can be specifically altered(for reviews on recombinogenic cloning see references [47–49]).Gateway cloning(Fig.2b)vectors have several features required for high-efficiency and accurate cloning using recombino-genic methods[10].Once an insert is cloned into an entry vector for Gateway cloning,it can be easily swapped into other Gateway vectors using clonase activity.This makes Gateway cloning very useful for applications where the target construct is required in various contexts like different solubility tags or different origins of replication.Ligation-independent cloning(Fig.2c)[50]addsflanking se-quence extensions to the target gene that are shorter than those used in recombinogenic methods while still avoiding some of the drawbacks of traditional cloning methods[51].By taking advan-tage of the controlled exonuclease activity of T4DNA polymerase, the LIC procedure creates long single-stranded overhangs of DNA in the vector and the insert.These overhangs hybridize on mixing the vector and insert together.When transformed into E.coli,the nicks are sealed by the cellular machinery.Hence no use of ligase is required,and since the vector has two fairly large overhangs which can be designed to be completely non-complementary to each other,the chances of re-sealing the vector are very small. Moreover,since this scheme does not require digestion of the in-sert with any restriction enzymes,there are no complications regarding multiple occurrences of the preferred cloning enzymes within the sequence to be cloned.A new cloning method calledTable1List of plasmid vectors.Vector name Fusion tags a Cleavage sites‘Scar’residues post-cleavage b Cloning method Promoter region Ori Res.Ref.Origins of replication and antibiotic resistance markers:pET11a T7None13amino acids Ligation T7ColE1Amp[99] pURI2-Ery6ÂHis EK protease M LIC lpp ColE1Ery[100] pACYCDuet-16ÂHis None13amino acids Ligation T7p15A Cmp[101] pT7LICK6ÂHis(Ct)None DHHHHHH(Ct)LIC T7pSC101Kan[12] pEC None N.A.N.A.Ligation P BAD pEC Kan[45] pMCSG216ÂHis TEV protease SNA LIC T7Clo-DF13Spec[102] pCOLADuet-16ÂHis None14amino acids Ligation T7ColA Kan[43] Cloning methods:pET32a Trx-6ÂHis EK protease AMA Ligation T7ColE1Amp[99] pVP168ÂHis-MBP TEV protease S Gateway T5ColE1Amp[8] pNIC28-Bsa46ÂHis TEV protease SM LIC T7ColE1Kan[4] pBL None N.A.N.A.SLiCE T7ColE1Amp[52] pCR-BluntII-None N.A.N.A.TOPO T7,SP6ColE1Kan,[55,56] TOPO ZeoTranscription promoters:pVP33A8ÂHis-MBP3C and TEV AIA Ligation T5ColE1Amp[8] pURI26ÂHis EK protease M LIC lpp ColE1Amp[13] pURI2-TEV6ÂHis TEV protease GM LIC lpp ColE1Amp[100] pURI36ÂHis EK protease M LIC T7ColE1Amp[13] pBAD/His6ÂHis EK protease DRWGSELE Ligation P BAD ColE1Amp[103] pCKSP6None N.A.N.A.Ligation SP6ColE1Amp[104] PinPoint Xa1Biotin Factor Xa None Ligation tac ColE1Amp[105] pCOLD I6ÂHis Factor Xa HM Ligation cspA ColE1Amp[72] Tag-switching plasmids:pHLIC6ÂHis TEV protease None LIC T7ColE1Amp[98] pHLIC-MBP6ÂHis-MBP TEV protease None LIC T7ColE1AmppMCSG76ÂHis TEV protease SNA LIC T7ColE1Amp[11] pMCSG96ÂHis-MBP TEV protease SNA LIC T7ColE1Amp[95] pMCSG106ÂHis-GST TEV protease SNA LIC T7ColE1Amp[102] pMCSG116ÂHis TEV protease SNA LIC T7p15A Cmp[102] pMCSG136ÂHis-MBP TEV protease SNA LIC T7p15A Cmp[97] pMCSG146ÂHis-GST TEV protease SNA LIC T7p15A Cmp[97] pLIC-His6ÂHis TEV protease GAAS LIC T7ColE1Amp[2] pLIC-MBP6ÂHis-MBP TEV protease GAAS LIC T7ColE1Amp[2] pLIC-GST6ÂHis-GST TEV protease GAAS LIC T7ColE1Amp[2] pOPINF6ÂHis3C protease GP LIC T7ColE1Amp[1] pOPINM6ÂHis-MBP3C protease GP LIC T7ColE1Amp[1] pOPINJ6ÂHis-GST3C protease GP LIC T7ColE1Amp[1] Protease cleavage sites:pMCSG19MBP-6ÂHis TVMV and TEV SNA LIC T7ColE1Amp[95] pVP33K8ÂHis-MBP3C and TEV AIA Ligation T5ColE1Kan[8] pCAL-n-EK CBP EK protease None LIC T7ColE1Amp[19] pHUE6ÂHis-Ub Usp2-cc None Ligation T7ColE1Amp[77] pHLIC-smt36ÂHis-SUMO SUMO protease None LIC T7ColE1AmpC-terminal His-tags:pT7LICB6ÂHis(Ct)None DHHHHHH(Ct)LIC T7ColE1Amp[12] pT7LICC6ÂHis(Ct)None DHHHHHH(Ct)LIC T7p15A Cmp[12] pT7LICK6ÂHis(Ct)None DHHHHHH(Ct)LIC T7pSC101Kan[12] pURI2-Cter6ÂHis(Ct)None HHHHHH(Ct)LIC lpp ColE1Amp[100]Ori,origin of replication;Res,antibiotic resistance marker;Ref,reference;Amp,ampicillin;His,histidine tag;EK,enterokinase;LIC,ligation independent coning;Ery, erythromycin;Cmp,chloramphenicol;Ct,C-terminal;Kan,kanamycin;N.A.,not applicable;TEV,Tobacco Etch Virus;Spec,spectinomycin;Trx,thioredoxin;MBP,maltose binding protein;Zeo,zeocin;3C,human rhinovirus3C protease site;GST,glutathione S-transferase;SUMO,Small Ubiquitin-like Modifier;TVMV,tobacco vein mottling virus protease;CBP,calmodulin binding protein.a All the fusion tags are on the N-terminus unless otherwise specified as Ct(C-terminus).b Extra N-terminal(unless otherwise specified)residues that remain after protease cleavage due to the design of the cloning site are listed in one letter amino acid codes.In some cases more residues may be required based on efficiency of protease cleavage.For vectors that have a tag but no cleavage site,the length of the tag is mentioned.V.Durani et al./Protein Expression and Purification85(2012)9–1711SLiCE(Seamless Ligation Cloning Extract)relies on in vitro recom-bination in bacterial cell extract(Fig.2d)[52].Extracts from the two recAÀstrains DH10B and JM109yielded the highest cloning efficiencies indicating that SLiCE is facilitated by a RecA-indepen-dent mechanism.SLiCE facilitates seamless cloning by recombining short end homologies(>15bp)with or withoutflanking heterolo-gous sequences.Seamless cloning using SLiCE has been demon-strated using plasmid vector pBL[52].Other recently-reported methods of cloning that fall into this category include Gibson clon-ing(enzymatic assembly of DNA molecules using T5exonuclease, Phusion polymerase and Taq ligase)[53]and CPEC(Circular Poly-merase Extension Cloning that involves extension of the overlap between single stranded vector and insert to form a complete plas-mid)[54].TOPO cloning(Fig.2e)is a method of cloning where a vector and insert with single nucleotide overhangs are ligated together by tak-ing advantage of topoisomerase activity[55].The single nucleotide overhang in the insert is created upon amplification by Taq poly-merase and this technique,like LIC,negates the use of restriction enzyme digest for the insert.The vector pCR-BluntII-TOPO(Invitro-gen)uses TOPO cloning in conjunction with the ccdB(control of cell death)gene for zero background cloning[56].The CcdB protein causes cell death by poisoning bacterial DNA gyrase,which leads to degradation of the host chromosome[57,58].Ligation of an in-sert into the vector disrupts the ccdB gene and enables the recom-binant colonies to grow.This method eliminates background from self-ligation of the vector.Transcription promotersPromoter regions affect the level of gene expression in E.coli,and the suitability of a promoter for protein expression depends on three main factors.First,the promoter must be strong and have the capacity to over-produce proteins to the extent that they be-come a significant percentage of the total expressed protein in the cell.Second,it is desirable to have a highly tunable(or at least highly repressible)promoter particularly for proteins that have adverse ef-fects on the growth of the host cell.Third,the promoter should be easily inducible typically by chemical or thermal means.Several promoter systems used for protein production in E.coli,their regu-lation mechanisms,and methods of induction can be found in refer-ences[59,60].Vectors with SP6(phage promoter,mainly used for cell free expression[61,62]),T5(phage promoter,induced by IPTG, very high level of expression,tight regulation possible[63]),hybrid lpp(E.coli promoter,induced by IPTG,very high level ofexpression Fig.2.Cloning schemes for(a)ligation-based cloning;(b)ligation-independent cloning;(c)recombinogenic cloning(Gateway);(d)SLiCE cloning;and(e)TOPO cloning.[64]),P BAD(E.coli promoter,induced by L-arabinose,variable level of expression from low to high,tight regulation possible[65]),tac (E.coli hybrid promoter,induced by IPTG,moderately high level of expression,high basal level of expression[66]),cspA(E.coli pro-moter,cold shock induction at temperatures below20°C,low level of expression[67])and T7(phage promoter,induced by IPTG,very high level of expression,tight regulation possible[68])promoters are listed in Table1.The pET vector system that has the promoter system from bacteriophage T7has been very widely used due to the orthogonality of this system to E.coli promoters,leading to ease of cloning toxic genes and also its capacity to overproduce proteins at very high levels constituting up to50%of the total cell protein [69–71].The T7promoter,along with some of the other promoters listed above,is indirectly induced by IPTG in that T7RNA polymer-ase is expressed from the lac promoter-operator.One of the limitations of the T7promoter system,however,is that rapid over-expression of relatively ill-behaved proteins can lead to pro-tein misfolding,aggregation and segregation into inclusion bodies. Although using a cold shock promoter like cspA[67](pCold vectors by Takara Bio Inc.[72])is sometimes a solution to this problem,a major disadvantage of that system is that it becomes repressed1–2h after lowering the temperature,and this time frame might not be sufficient for a high level accumulation of all proteins.Fusion tagsBesides cold shock,there are several approaches to the expres-sion of proteins that end up in the insoluble fraction,including the co-overexpression of chaperones[23–25],periplasmic expres-sion of proteins[73]and the use of solubilizing fusion tags.Protein fusions are efficient tools for protein purification,detection and immobilization.One of the most commonly used fusion tags for protein purification is the hexahistidine(6ÂHis)[13]tag that facil-itates purification by immobilized metal-ion affinity chromatogra-phy(IMAC)purification with Ni–NTA agarose.This method is inexpensive;the resin has very high capacity and can be reused many times.It is less expensive than other resins such as streptavi-din agarose used for purification of biotin-tagged proteins,and it has a higher capacity than the less-expensive amylose resin used to purify proteins with an MBP tag[59,74].Although fusion tags were originally developed for purification purposes,it was found that many tags lead to better solubilization of proteins fused at their C-termini.It is believed that the general mechanism for this effect involves the fast and efficient folding of the N-terminal fusion tag upon exiting the ribosome immediately after translation,thus pulling the C-terminally fused protein into solution.The most com-monly used fusion tags for solubility enhancement are maltose binding protein(MBP)[14,15],calmodulin-binding peptide(CBP) [18,19],glutathione S-transferase(GST)[16,17],and thioredoxin (Trx)[20].More recently it has been shown that the N-terminal fu-sion of SUMO protein[75,76],ubiquitin[77],and HaloTag7[78]also enhance expression levels and solubility of ill-behaved proteins.Several fusion tags can have multiple functionalities;for instance, MBP and HaloTag7can be used for both solubility enhancement and purification.Similarly,SUMO and ubiquitin(Ub)protein fusions can be used as both solubility tags and also protease cleavage tags.Some-timesfinding the optimum tag for an application requires some trial and error and also some applications may require the same protein with different tags.Hence,tag switching vectors where the same in-sert can be used to clone with various tags are desirable.Protease cleavage sitesEven though fusion tags often solubilize proteins and provide options for easy purification,it is often desirable to remove them after purifimon proteases for this purpose include TEV protease[79–81]and related3C protease[82],enterokinase [83–85],Factor Xa[86],thrombin[87,88]and SUMO protease [76].TEV protease,the capsid protease of the tobacco etch virus, is among the most robust of the proteases in common use[89]. It is more specific than enterokinase[85]or Factor Xa[90],and it is active under a wide range of conditions.It is commercially avail-able and can also be overexpressed and purified from E.coli using a standard protocol[91].The general form of its recognition se-quence is taken to be E-X-X-Y-X-Q-(G/S),with cleavage after the Gln residue;the most commonly used sequence is ENLYFQG.Re-cently,Kapust et al.demonstrated that the TEV protease will cleave with greater than90%efficiency with12different amino acids at the P10site(C-terminal to Gln)and greater than50%efficiency for all other amino acids except proline[92].Effectively,TEV pro-tease can be used to quantitatively produce mature proteins with any N-terminal amino acid except Pro under reasonable reaction conditions.However,proteins with structured N-terminal regions sometimes do not display very efficient cleavage with TEV protease presumably due to inaccessibility of the protease recognition and cleavage site.For such cases,having an alternative option is desir-able,such as Factor Xa,which cleaves I-(E/D)-G-R-X between R and X,where X can be any amino acid except proline[93].Another op-tion is the SUMO protein fusion.In this case,the100residue SUMO protein serves as the recognition site for SUMO protease,which cleaves after the C-terminal Gly of SUMO.It has been shown that recombinant SUMO–GFP fusions are efficiently cleaved when any amino acid except proline is in P1´position of the cleavage site [76].Similar to SUMO,the ubiquitin tag also serves as a recognition site for deubiquitylating enzymes(DUBs),however most DUBs that have been isolated from various species are relatively large and dif-ficult to express and purify.No stable DUB known with general activity against a range of fusion proteins was known.Recently,a mouse DUB,Usp2,was engineered to provide a minimal catalyti-cally active deubiquitylating domain(Usp2-cc)[77].It was ex-pressed and purified with a polyhistidine tag that allowed the in vitro cleavage of Ub from the desired protein as well as its selec-tive removal from the cleavage reaction.Seamless cloning and traceless taggingGeneration of the overhang for cloning into a vector requires restriction enzyme scission,which places a limitation on the se-quence of the overhang and usually results in a particular protein sequence at the site of fusion.A way to get around this problem and accomplish‘seamless’cloning[94]is to digest with a type IIs restriction enzyme which cuts outside of its recognition sequence and therefore in any sequence.(GeneArt by Invitrogen and CloneEZ by GenScript are kits that can be used for seamless cloning.)Even for routine protein expression and purification,it would be useful to have a veryflexible plasmid vector for high-level E.coli overex-pression of proteins using an N-terminal6ÂHis tag that could be removed tracelessly–which is to say,with no residues left behind and few or no constraints on the amino acid next to the scissile bond.To circumvent some issues with ligation-dependent cloning, a ligation-independent method is desirable,but with minimal ex-pense,and with theflexibility to use ligation-dependent methods, as well.Several vectors which nearlyfit these criteria are available, perhaps most notably the pMCSG7vector[11]and the pHLIC vec-tor system(described here).Vectors for traceless tagging–some examplesToday several plasmid vectors are available that have cloning sites designed to attach fusion tags and also provide for proteaseV.Durani et al./Protein Expression and Purification85(2012)9–1713cleavage sites to remove the fusion tags later.These vectors have combinations of different methods of cloning,different fusion tags and different protease cleavage sites (several examples are listed in Table 1).For instance,vectors pURI [13]and pCAL-n-EK [19]can be used for ligation-independent cloning,and use enterokinase,which is not as specific as TEV and also more expensive.Vectors like pOPINJ [1],pLIC [2],pNIC28[4],pVp [8]allow for TEV cleavage of 6ÂHis tag,optionally C-terminal)6ÂHis tag to a target protein cloned by LIC under the control of the T7promoter.Variants of this vector that allow for TEV cleavable MBP and GST fusions are also available and this series has some ColE1derived vectors and others with p15A derived replicons for co-expression [97].However,the LIC cloning scheme designed for these vectors requires that the cleaved N-terminal sequence begin with Ser-Asn-Ala.We have constructed a related vector in which there are no constraints on the N-terminal sequence,and in which the protease recognition se-quence can be removed or changed by a simple standard cloning step.We call the resulting vector pHLIC (plasmid for 6ÂHis tagging and Ligation Independent Cloning,Fig.3and Supplementary material ).To achieve high-level expression and simple,inexpensive purifi-cation,we selected a pET vector system with a T7promoter for overexpression in DE3-lysogenized cells,and we selected an N-ter-minal 6ÂHis-tag.It would be difficult to achieve ‘‘traceless’’scission with a C-terminal tag,since virtually all useful (highly specific and active)proteases have their recognition elements upstream of or overlapping the scissile bond.To truly make this traceless,we se-lected the robust and easy-to-prepare TEV protease,which cleaves Fig.3.Schematic maps of the pHLIC and pHLIC-MBP plasmids.The AlwNI site is not unique in the pHLIC-MBP vector.4.LIC scheme for pHLIC.BsaI digested pHLIC vector (a)is treated with T4DNA polymerase in the presence of dGTP to create large overhangs (b).The PCR product for insert (c)is treated with T4DNA polymerase in the presence of dCTP to create complementary overhangs (d).Then (b)and (d)are mixed together leading to the formation desired clone by hybridization of the overhangs of the vector and insert.This product has nicks,at positions indicated by arrows in (e),which are resolved in the cell upon transformation.and Purification 85(2012)9–17。